There has been known a microscope that irradiates an observation plane of a sample locally with light and that receives fluorescence radiated from the sample. The microscope, with such an arrangement, allows an observation of sectioned planes of a sample. Such a microscope, which is capable of preventing light from illuminating that portion of a sample which is not an observation plane, can reduce the background during the observation of the sample and can also reduce, for example, harmful action to a sample or attenuation of fluorescence that are caused by irradiation of the sample with excitation light.
Patent Literatures 1 and 2 and Non Patent Literature 1 each disclose a highly inclined and laminated optical sheet microscopy for an optical microscope, with which technique illumination light is refracted with use of an objective lens to irradiate a sample in an oblique direction with respect to the optical axis of the objective lens. FIG. 18 shows enlarged views of part of an optical microscope that uses the highly inclined laminated optical sheet microscopy of Patent Literature 1. In a case where an optical microscope is arranged such that illumination light enters a sample in a direction so angled as to be close to perpendicular to the optical axis of the objective lens as illustrated in FIG. 18, the optical microscope can irradiate a sample with a thin layer-shaped light having a small thickness along the direction of the optical axis of the objective lens. According to Patent Literature 1, the optical microscope is capable of continuously capturing an image of a sample while moving the focal position of the objective lens to produce a three-dimensional image at a high resolution. Patent Literature 1 discloses in particular that the optical microscope is capable of imaging at a single-molecule level.
The technique disclosed in Patent Literature 1 may serve to adjust the position on an objective lens at which position illumination light enters the objective lens, so that the angle θ between optical axes of illumination light refracted and the objective lens is close to 90 degrees. The technique, however, does not allow the angle θ to be exactly 90 degrees. Thus, the optical axis of illumination light is not parallel to the observation plane of the objective lens. This arrangement results in an image being ununiformly out of focus to varying degrees over the observation plane, the image thus having decreased quality evenness. Further, the above arrangement lets illumination light illuminate a region of a sample which region is other than the observation plane, and thus increases the background, with the result of a fluorescence image having a decreased resolution. In particular, in a case of producing a three-dimensional image, such an image will have a low resolution along the direction parallel to the optical axis of the objective lens.
Non Patent Literature 2 discloses SPIM (selective plane illumination microscope), which irradiates a sample with a thin layer-shaped sheet light, collects radiated fluorescence with use of an objective lens having an optical axis perpendicular to the optical axis of the sheet light, and forms an image of the collected fluorescence with use of a camera. In a case of observing a sample with use of the SPIM of Non Patent Literature 2, the SPIM rotates agarose gel in which the sample is embedded and thus irradiates the sample with sheet light from various angles, and captures an image of the sample with use of a camera. FIG. 19 is a diagram schematically illustrating a part of the microscope of Non Patent Literature 2 on which part a sample is placed. Patent Literature 3 discloses that in a case of collecting light with use of an illumination lens and irradiating a sample with the collected light in the form of layer-shaped sheet light, the sheet light has a thickness along the direction parallel to the optical axis of the objective lens which thickness depends on the numerical aperture of the illumination lens. This disclosure indicates that in a SPIM, increasing the numerical aperture of the illumination lens can reduce the thickness of sheet light and can thereby reduce the background.
Patent Literatures 4 to 6 each similarly disclose a microscope that collects fluorescence from a sample with use of an objective lens having an optical axis perpendicular to the optical axis of sheet light and that thereby produces a three-dimensional image of the sample.
In a case of irradiating a sample with sheet light in the direction perpendicular to the optical axis of an objective lens as with the microscopes disclosed in Patent Literatures 3 to 6 and Non Patent Literature 2, it is possible to reduce the background and, as a result, obtain a high resolution.